Fig 1.
The anticancer activity of diethyldithiocarbmamate (DDC) is dependent on copper.
(A) Disulfiram is metabolized to diethyldithiocarbamate (DDC) and DDC complexes with Copper (Cu) (II). (B) Cytotoxicity curves for DSF (●) and DSF + CuSO4 (■) were obtained with the IN CELL Analyzer using U87 glioblastoma cells where cell viability was assessed based on loss of plasma membrane integrity 72 hours following treatment; i.e. total cell count and dead cell count were determined using Hoechst 33342 and ethidium homodimer staining, respectively. (C) Cytotoxicity curves for DDC (●) and DDC + CuSO4 (■); where cytotoxicity was measured as described above. (D) DDC and Cu(DDC)2 IC50 for U251, MDA-231-BR, and A549 cancer cell lines as well as HBEcP a normal cell line; averages (±SEM) are reported from three separate experiments each done in triplicate. (E) Photograph of DDC, CuSO4 and Cu(DDC)2 solutions in water.
Fig 2.
Diethyldithiocarbamate (DDC) loading into DSPC/Chol (55:45) liposomes prepared with encapsulated 300 mM CuSO4.
(A) Photograph of solutions consisting of DDC (5mg/mL) and added to CuSO4-containing DSPC/Chol (55:45) liposomes (20 mM liposomal lipid) over a 1 hour at 25°C. (B) Formation of Cu(DDC)2 inside DSPC/Chol liposomes (20 mM) as a function of time over 1 hour at 4(●), 25(■) and 40(▲)°C following addition of DDC at a final DDC concentration of (5 mM); Cu(DDC)2 was measured using a UV-Vis spectrophotometer and lipid was measured using scintillation counting. (C) Cu(DDC)2 formation inside DSPC/Chol (55:45) liposomes over time where the external pH was 7.4 (▲) and 3.5 (▼). (D) Measured Cu(DDC)2 as a function of increasing DDC added, represented as the theoretical Cu(DDC)2 to total liposomal lipid ratio; where the lipid concentration was fixed at 20 mM and final DDC concentration was varied. (E) Cryo-electron microscopy photomicrograph of CuSO4- containing DSPC/Chol (55:45) liposomes and the same liposomes after formation of encapsulated Cu(DDC)2. (F) Size of the CuSO4- containing liposomes and liposomes with encapsulated Cu(DDC)2 as determined by quasi-electric light scattering and cryo-electron microscopy; data points are given as mean ± SD.
Fig 3.
Characterization of copper-complex loading method.
(A) Measured (AAS) copper to liposomal lipid ratio (black bars) compared to measured Cu(DDC)2 (UV-Vis spectrophotometer) to liposomal lipid ratio (grey bars) after DDC was added to CuSO4-containing DSPC/Chol liposomes prepared with different amounts of DSPE-PEG2000 (ranging from 0 to 5 mole%). (B) Formation of Cu(DDC)2 inside CuSO4-containing DSPC/Chol liposomes as a function of the CuSO4 concentration used to prepare the liposomes (ranging from 0 to 300 mM); where the measured copper (AAS) to liposomal lipid ratio (black bar) is compared to the measured Cu(DDC)2 (UV-Vis spectrophotometer) to liposomal lipid ratio (grey bar). (C) Linear regression analysis comparing measured (AAS) copper concentration (assuming encapsulated copper was free in solution) to measured Cu(DDC)2 (UV-Vis spectrophotometer) concentration (assuming encapsulated Cu(DDC)2 was free in solution); R2 = 0.9754; each data point represents a mean ± SEM determined from at least three separate experiments done in duplicate.
Fig 4.
Donor systems that can be used in Copper(II)-complex loading.
Copper is able to form complexes with compounds containing S-Donor, O-Donor and N,O-Donor systems as well as other mixed donor systems. Examples of drugs that are described here, in addition to DDC (an S-Donor), include Quercetin (Qu) (an O-Donor), Clioquinol (CQ) (an N,O donor) as well as CX-5461, previously not identified as a copper complexing agent. Each was loaded into DSPC/Chol (55:45 mol ratio) liposomes prepared with 300 mM CuSO4. The loading temperature used in these examples was 25, 50, 40 and 60°C, respectively.
Fig 5.
Preliminary tolerability and plasma elimination profiles for liposomal formulations of Cu(DDC)2, Cu(CQ)2, CuQu and CuCX-5461 after intravenous injection into CD-1 mice.
Mice were injected with a single dose of 15 mg/kg Cu(DDC)2 (-●-), 30mg/kg Cu(CQ)2 (-■-), 70mg/kg CuQu (-▲-) and 50 mg/kg Cu-CX-5461 (-▼-). (A) Changes in body weight following administration of the indicated liposomal formulation where body weights were measured over 14 days after injection (n = 3). (B) Preliminary plasma elimination profiles of the indicated liposomal formulations where the copper-complexed compound was measured at 1, 4, 8 and 24 hrs after administration (n = 4); concentrations were measured using HPLC or AAS as described in the Methods.